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Engineering a cell’s regulatory networks to dynamically control gene expression has been considered a new frontier in biological engineering. In cyanobacteria, the lack of well-characterized, modular gene regulatory elements makes regulatory network engineering challenging. Here, we suggest potential tools to modify various gene expression steps in cyanobacterial regulatory networks. Engineering a cell’s regulatory networks to dynamically control gene expression has been considered a new frontier in biological engineering. In cyanobacteria, the lack of well-characterized, modular gene regulatory elements makes regulatory network engineering challenging. Here, we suggest potential tools to modify various gene expression steps in cyanobacterial regulatory networks.

The invention of the Kalman filter is a crowning achievement of filtering theory—one that has revolutionized technology in countless ways. By dealing effectively with noise, the Kalman filter has enabled various applications in positioning, navigation, control, and telecommunications. In the emerging field of synthetic biology, noise and context dependency are among the key challenges facing the successful implementation of reliable, complex, and scalable synthetic circuits. Although substantial further advancement in the field may very well rely on effectively addressing these issues, a principled protocol to deal with noise—as provided by the Kalman filter—remains completely missing. Here we develop an optimal filtering theory that is suitable for noisy biochemical networks. We show how the resulting filters can be implemented at the molecular level and provide various simulations related to estimation, system identification, and noise cancellation problems. We demonstrate our approach in vitro using DNA strand displacement cascades as well as in vivo using flow cytometry measurements of a light-inducible circuit in Escherichia coli.

Engineering biological processes has become a standard approach to produce various commercially valuable chemicals, therapeutics, and biomaterials. Among these products, bacterial cellulose represents major advances to biomedical and healthcare applications. In comparison to properties of plant cellulose, bacterial cellulose (BC) shows distinctive characteristics such as a high purity, high water retention, and biocompatibility. However, low product yield and extensive cultivation times have been the main challenges in the large-scale production of BC. For decades, studies focused on optimization of cellulose production through modification of culturing strategies and conditions. With an increasing demand for BC, researchers are now exploring to improve BC production and functionality at different categories: genetic, bioprocess, and product levels as well as model driven approaches targeting each of these categories. This comprehensive review discusses the progress in BC platforms categorizing the most recent advancements under different research focuses and provides systematic understanding of the progress in BC biosynthesis. The aim of this review is to present the potential of ‘modern genetic engineering tools’ and ‘model-driven approaches’ on improving the yield of BC, altering the properties, and adding new functionality. We also provide insights for the future perspectives and potential approaches to promote BC use in biomedical applications.

Plant synthetic biology holds great promise for engineering plants to meet future demands. Genetic circuits are being designed, built and tested in plants to demonstrate the proof of concept. However, developing these components in monocots, which the world relies on for grain, lags behind dicot models, such as Arabidopsis thaliana and Nicotiana benthamiana. Here, we show the successful adaptation of a ligand‐inducible sensor to activate an endogenous anthocyanin pathway in the C4 monocot model Setaria viridis. We identify two transcription factors that can be expressed as a single transcript that are sufficient to induce endogenous anthocyanin production in S. viridis protoplasts and whole plants in a constitutive or ligand‐inducible manner. We also test multiple ligands to overcome physical barriers to ligand uptake, identifying triamcinolone acetonide (TA) as a highly potent inducer of this system. Using hyperspectral imaging and a discriminative target characterisation method in a near‐remote configuration, we can non‐destructively detect anthocyanin production in leaves in response to ligands. This work demonstrates the use of inducible expression systems in monocots to manipulate endogenous pigmentation production for remote detection. Applying inducible anthocyanin production coupled with sensitive detection algorithms could enable crop plants to report on the status of field contamination or detect undesirable chemicals impacting agriculture, ushering in an era of agriculture‐based sensor systems.

Phenotypic variation is the raw material of adaptive Darwinian evolution. The phenotypic variation found in organismal development is biased towards certain phenotypes, but the molecular mechanisms behind such biases are still poorly understood. Gene regulatory networks have been proposed as one cause of constrained phenotypic variation. However, most pertinent evidence is theoretical rather than experimental. Here, we study evolutionary biases in two synthetic gene regulatory circuits expressed in Escherichia coli that produce a gene expression stripe—a pivotal pattern in embryonic development. The two parental circuits produce the same phenotype, but create it through different regulatory mechanisms. We show that mutations cause distinct novel phenotypes in the two networks and use a combination of experimental measurements, mathematical modelling and DNA sequencing to understand why mutations bring forth only some but not other novel gene expression phenotypes. Our results reveal that the regulatory mechanisms of networks restrict the possible phenotypic variation upon mutation. Consequently, seemingly equivalent networks can indeed be distinct in how they constrain the outcome of further evolution. Synopsis Analyses in synthetic circuits show that mutations result in distinct novel phenotypes in two circuits that showed the same phenotype before mutation. This constrained phenotypic variation is caused by differences in the circuits’ regulatory mechanisms. Two synthetic circuits expressed in E. coli that produce the same phenotype, but through different regulatory mechanisms, are used to study the molecular mechanisms underlying constrained phenotypic variation during evolution. The two networks create different spectra of novel phenotypes after mutation. A combination of experimental measurements, mathematical modeling and DNA sequencing shows that the regulatory mechanisms restrict the phenotypic variation that becomes accessible upon mutation. Graphical Abstract Analyses in synthetic circuits show that mutations result in distinct novel phenotypes in two circuits that showed the same phenotype before mutation. This constrained phenotypic variation is caused by differences in the circuits’ regulatory mechanisms.

Auxin-regulated transcription pivots on the interaction between the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressor proteins and the AUXIN RESPONSE FACTOR (ARF) transcription factors. Recent structural analyses of ARFs and Aux/IAAs have raised questions about the functional complexes driving auxin transcriptional responses. To parse the nature and significance of ARF–DNA and ARF–Aux/IAA interactions, we analyzed structure-guided variants of synthetic auxin response circuits in the budding yeast Saccharomyces cerevisiae. Our analysis revealed that promoter architecture could specify ARF activity and that ARF19 required dimerization at two distinct domains for full transcriptional activation. In addition, monomeric Aux/IAAs were able to repress ARF activity in both yeast and plants. This systematic, quantitative structure-function analysis identified a minimal complex—comprising a single Aux/IAA repressing a pair of dimerized ARFs—sufficient for auxin-induced transcription.

Background Frame-shifted genes results in non-functional peptides. Because of this complete loss of function, frame-shifted genes have never been used in constructing synthetic gene circuits. Results Here we report that the function of gene circuits is rescued by a frame-shifted gene, which functions by translating from a non-natural start codon. We report a single nucleotide deletion mutation that developed in the λ-repressor cI within a synthetic genetic NOT gate in Escherichia coli during growth and through this mutation, a non-functional synthetic gene circuit became functional. This mutation resulted in a frame-shifted cI, which showed effective functionality among genetic NOT-gates in Escherichia coli with high regulatory ranges (> 300) and Hill coefficient (> 6.5). The cI worked over a large range of relative copy numbers between the frame-shifted gene and its target promoter. These properties make this frame-shifted gene an excellent candidate for building synthetic gene circuits. We hypothesized a new operating mechanism and showed evidence that frame-shifted cI was translated from non-natural start codon. We have engineered and tested a series of NOT gates made from a library of cI genes, each of which starts from a different codon within the first several amino acids of the frame-shifted cI. It is found that one form with start codon ACA, starting from the 3rd codon had similar repression behavior as the whole frame-shifted gene. We demonstrated synthetic genetic NAND and NOR logic-gates with frame-shifted cI. This is the first report of synthetic-gene-circuits made from a frame-shifted gene. Conclusions This study inspires a new view on frame-shifted gene and may serve as a novel way of building and optimizing synthetic-gene-circuits. This work may also have significance in the understanding of non-directed evolution of synthetic genetic circuits.

The processes that keep a cell alive are constantly challenged by unpredictable changes in its environment. Cells manage to counteract these changes by employing sophisticated regulatory strategies that maintain a steady internal milieu. Recently, the antithetic integral feedback motif has been demonstrated to be a minimal and universal biological regulatory strategy that can guarantee robust perfect adaptation for noisy gene regulatory networks in Escherichia coli. Here, we present a realization of the antithetic integral feedback motif in a synthetic gene circuit inmammalian cells. We show that the motif robustly maintains the expression of a synthetic transcription factor at tunable levels even when it is perturbed by increased degradation or its interaction network structure is perturbed by a negative feedback loop with an RNA-binding protein. We further demonstrate an improved regulatory strategy by augmenting the antithetic integral motif with additional negative feedback to realize antithetic proportional–integral control. We show that this motif produces robust perfect adaptation while also reducing the variance of the regulated synthetic transcription factor. We demonstrate that the integral and proportional–integral feedback motifs can mitigate the impact of gene expression burden, and we computationally explore their use in cell therapy. We believe that the engineering of precise and robust perfect adaptation will enable substantial advances in industrial biotechnology and cell-based therapeutics.

Membrane systems are used increasingly for water treatment, recycling water from wastewater, during food processing, and energy production. They thus are a key technology to ensure water, energy, and food sustainability. However, biofouling, the build-up of microbes and their polymeric matrix, clogs these systems and reduces their efficiency. Realizing that a microbial film is inevitable, we engineered a beneficial biofilm that prevents membrane biofouling, limiting its own thickness by sensing the number of its cells that are present via a quorum-sensing circuit. The beneficial biofilm also prevents biofilm formation by deleterious bacteria by secreting nitric oxide, a general biofilm dispersal agent, as demonstrated by both short-term dead-end filtration and long-term cross-flow filtration tests. In addition, the beneficial biofilm was engineered to produce an epoxide hydrolase so that it efficiently removes the environmental pollutant epichlorohydrin. Thus, we have created a living biofouling-resistant membrane system that simultaneously reduces biofouling and provides a platform for biodegradation of persistent organic pollutants.

Nicotiana benthamiana has emerged as a premier plant biofactory for recombinant protein and metabolite production due to its high metabolic versatility, ease of cultivation and permissiveness to transient expression vectors. However, challenges such as transgene silencing, low yields and metabolic toxicity limit its scalability. Synthetic gene circuits offer transformative solutions by enabling precise transgene expression control, dynamic signal processing and metabolic pathway optimization. Recent advancements include novel sensor systems responsive to chemical and electromagnetic signals, synthetic promoters integrated with programmable transcription factors and virus‐derived replicons for transcriptional signal amplification. Recombinase‐based processors further enhance conditional gene expression and cellular memory capabilities. Innovative platforms like plant cell packs and self‐sustained bioluminescence systems facilitate rapid prototyping of gene circuits, enabling high‐throughput screening and optimization. Future strategies focus on stable genomic integration, positional effect mitigation and accelerated transgenic line generation using morphogenic regulators and CRISPR systems. By addressing these challenges, synthetic biology can unlock the full potential of N. benthamiana as a scalable and sustainable biofactory for molecular farming, biosensing and advanced bioproduction applications.